There are many solutions to do one thing, and different solutions have different protocols. Then back to the source, what solution should I choose? How to solve the problem when you encounter it?
The most common problems encountered in CRISPR-Cas9 gene editing are: not picking a single clone, unsuccessful editing/efficiency . I believe that many CRISPR novices, including me, will focus more on sgRNA design, infection/transfection, and single-clone selection, until the final gene knockout efficiency is still very low or unsuccessful. The reason
will first be attributed to the low efficiency of sgRNA , so it will choose to do it again.
At the same time, the name of CRISPR-Cas9 is also very confusing, which will let us subconsciously put the two elements of CRISPR-Cas9.
However, CRISPR-Cas9 is only the first half, and the second half is DNA repair .
This means that even if the efficiency of sgRNA is very high, everything is perfect, but if DNA repair goes wrong, all previous efforts will be lost.
Therefore, don't forget the important role of DNA repair in gene editing.
At the stage of experimental design, we must fully consider the purpose of the subject and select the corresponding DNA repair method.
From the literature, we can see that under the stimulation of external damage, such as intracellular reactive oxygen species, ionizing radiation, ultraviolet damage, and drug intervention, etc., cells can initiate 7 repair pathways to deal with different types of damage:
(1) Direct repair (DR) pathway;
(2) Base excision repair (BER);
(3) Nucleotide excision repair (NER);
(4) Base error Mismatch repair (mismatch repair, MMR) to correct base mismatches;
(5) homologous repair (HR);
(6) non-homologous end-joining (NHEJ) pathway;
( 7) translesion DNA synthesis (translesion DNA synthesis, TLS);
Among them, the post-HR and NHEJ pathways specialize in repairing DNA double-strand breaks (DSBs).
The early principle of CRISPR-Cas9 is to induce DNA double bond breaks (DSBs), and then rely on DNA repair to complete gene editing.
Although NHEJ and HDR are still the mainstream for gene repair in CRISPR, the method of microhomology-mediated end-joining (MMEJ) has also been active recently .
A brief diagram of these three DNA repair methods is as follows:
3. DSB repair method in CRISPR-Cas9
3.1 HEAVEN
Non-homologous end joining (NHEJ) or classical non-homologous end joining (C-NHEJ) is the main cell repair pathway for DSB repair by most eukaryotes .
It occurs in all stages of the cell cycle and is generally considered a rapid repair mechanism (approximately 10 minutes).
In the most basic sense, the working principle of this mechanism is to reconnect the blunt ends of DNA through a small process.
Specific steps are as follows:
(1) After the formation of DSBs, p-γH2AX and ATM are sheathed at both ends of DSBs, while Ku70/80 is sheathed in order, followed by DNA-PKcs sheathing to form a continuous sheath, and the blunt end of DSBs is pulled through DNA-PKcs Recently, it appeared that Artemis protein jammed both ends of DSBs:
(2) Afterwards, under the action of a variety of enzymes, DNA repair remains as before.
NHEJ has the following characteristics:
(1) NHEJ-mediated DSBs repair often causes indels errors, so a null, knockout effect is often achieved. Indels errors generated by NHEJ during the repair process are usually very small (1-10 bp), but the difference is huge, so the probability of frameshift mutations is about 2/3 .
(2) NHEJ-mediated DSBs repair does not necessarily introduce indels, but the DSBs without nucleotide damage caused by Cas9 are only about 5%; even if indels are not introduced after NHEJ repair, the target area of such precise repair will be repeated again It is cut by Cas9, but indels products will not. Since the time required for the division of a single cell and the DNA repair cycle is less than 1 hour, the process of introducing DSBs by Cas9 into DSBs and then repairing by NHEJ will be repeated in cycles. As a result, indels will be introduced on most chromosomes within a day . This shows that CRISPR-Cas9, which relies on the NHEJ repair method, is fast, efficient and high . Our previous series of protocols chose this method.
(3) It should be noted that Cas9 may not fall off the DNA immediately after causing DSBs, which will also hinder the NHEJ repair of DNA.
(4) NHEJ combined with paired sgRNA can also achieve the effect of knocking out the whole segment.
(5) NHEJ is active throughout the cell cycle, and its activity increases from G1 phase to G2/M, namely: G1 <S <G2/M.
3.2 HDR
If NHEJ is an error-prone DNA repair method, we take advantage of the error-prone characteristics of NHEJ to introduce indels. More often, we hope to achieve precise repair results. For example , diseases such as sickle-cell anemia ( SCA ) have been identified because of related gene mutations. If gene editing technology can be used, the changes can be accurately edited. The mutation site can achieve the therapeutic effect of drawing money from the bottom of the tank.
Currently, the CRISPR/Cas9 therapy CTX001 developed by CRISPR Therapeutics for the treatment of β-thalassemia has entered the clinical trial stage.
This precise editing also relies on the precise repair of DNA repair, that is, homologous repair (HR; Homology Directed Repair, HDR).
HDR is the second most common DSB repair mechanism in eukaryotes (except for budding yeast, which is mainly HDR). Unlike NHEJ, HDR relies on homologous repair templates (usually sister chromatids) to repair broken DNA.
HDR is active in late S and G2 stages. The HDR mechanism can be used for CRISPR-Cas9-mediated gene editing by introducing an exogenous DNA template that contains the homologous sequence of the target gene .
The following diagram briefly introduces the steps of accurately inserting sequences into the genome using the CRISPR-Cas9 method via HDR.
HDR has the following characteristics:
(1) HDR requires the template to have a sufficiently long homology arm sequence;
(2) HDR is active in the late S and G2 phases;
(3) It is necessary to pay attention to the design of repair templates, ssDNA templates (ssODNs for short) are usually used for smaller Gene modification, small insertion/editing may only need 30-50 bp homology arm, but this length is not absolute, usually 50-80 bp homology arm is used;
3.3 MMEJ
Microhomology - mediated end - joining (MMEJ) Compared with NHEJ and HDR, MMEJ has not received much attention.
MMEJ has the following characteristics
(1) The required homology arm is shorter, 5-25 bp;
(2) MMEJ is more active in the M phase and early S phase;
Comparison of three DSB repair methods
(1) MMEJ is not as perfect and precise repair as HDR, but it is easier to predict than NHEJ. The short homologous regions (5-25 bp) on both sides of DSB can be located to the target DNA of gene editing Sequence, that is, accuracy: HDR> MMEJ> NHEJ.
(2) But for species without a good HDR system, even if there is a repair template, the repair method is still NHEJ;
(3) The longer the homology of HDR, the higher the recombination efficiency, but the longer the homology arm, the longer the cloning time. The longer. In contrast, the short homologous sequence of MMEJ is easier to obtain by PCR amplification. Efficiency and simplicity: NHEJ> MMEJ> HDR;
(4) Although NHEJ is highly efficient, it is error-prone; HDR has the highest accuracy but the lowest efficiency; therefore, it can be knocked in, replaced or deleted if the activity of MMEJ is allowed. The effect of nucleotides, but the efficiency is lower than NHEJ;
4. The effect of cell cycle on DNA repair
In the above, we have repeatedly mentioned the active time periods of different DNA repair methods in the cell cycle. For example, NHEJ is active in the whole cycle and is more active with the recommendation of the cell cycle; HDR is active in the terminal S and G2 phases, and MMEJ is complementary Sexually active in the M and S phases.
In particular, changes in the cell cycle can also affect the efficiency of gene editing.
For example, in the 2018 Nature Medicine article CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response ,
(1) In conventional knockout of human retinal pigment epithelial cells (RPE1), Cas9-induced DSBs activate the TP53 gene The activation of the TP53 gene makes the cell stay in the G1 phase, so the NHEJ activity is up-regulated and the HDR activity is down-regulated (as shown in the figure below);
(2) After knocking out the TP53 gene, the G1 cycle arrest caused by TP53 activation is abolished, making The cell cycle continues to operate normally, so the activity of HDR is maintained.
The final conclusion is that the lack of TP53 will promote HDR-mediated CRISPR-Cas9, the reason is cell cycle influence. The icons are as follows:
This article attempts to compare the mechanisms and characteristics of different DSB repair methods, hoping to deepen my understanding of CRISPR-Cas9, and further understand the CIRSPR-Cas9 scheme design and the issues that need to be considered when debugging is required for experimental failure.
Of course, if you have any questions, you can discuss it together in the next live broadcast on March 2, 2021!
